Kinetics of γH2AX and phospho-histone H3 following pulse treatment of TK6 cells provides insights into clastogenic activity.

The desire for in vitro genotoxicity assays to provide higher information content, especially regarding chemicals' predominant genotoxic mode of action, has led to the development of a novel multiplexed assay available under the trade name MultiFlow®. We report here on an experimental design variation that provides further insight into clastogens' genotoxic activity. First, the standard MultiFlow DNA Damage Assay-p53, γ H2AX, phospho-histone H3 was used with human TK6 lymphoblastoid cells that were exposed for 24 continuous hours to each of 50 reference clastogens. This initial analysis correctly identified 48/50 compounds as clastogenic. These 48 compounds were then evaluated using a short-term, 'pulse' treatment protocol whereby cells were exposed to test chemical for 4 h, a centrifugation/washout step was performed, and cells were allowed to recover for 20 h. MultiFlow analyses were accomplished at 4 and 24 h. The γ H2AX and phospho-histone H3 biomarkers were found to exhibit distinct differences in terms of their persistence across chemical classes. Unsupervised hierarchical clustering analysis identified three groups. Examination of the compounds within these groups showed one cluster primarily consisting of alkylators that directly target DNA. The other two groups were dominated by non-DNA alkylators and included anti-metabolites, oxidative stress inducers and chemicals that inhibit DNA-processing enzymes. These results are encouraging, as they suggest that a simple follow-up test for in vitro clastogens provides mechanistic insights into their genotoxic activity. This type of information will contribute to improve decision-making and help guide further testing.

Comparison of in vitro and in vivo clastogenic potency based on benchmark dose analysis of flow cytometric micronucleus data.

The application of flow cytometry as a scoring platform for both in vivo and in vitro micronucleus (MN) studies has enabled the efficient generation of high quality datasets suitable for comprehensive assessment of dose-response. Using this information, it is possible to obtain precise estimates of the clastogenic potency of chemicals. We illustrate this by estimating the in vivo and the in vitro potencies of seven model clastogenic agents (melphalan, chlorambucil, thiotepa, 1,3-propane sultone, hydroxyurea, azathioprine and methyl methanesulfonate) by deriving BMDs using freely available BMD software (PROAST). After exposing male rats for 3 days with up to nine dose levels of each individual chemical, peripheral blood samples were collected on Day 4. These chemicals were also evaluated for in vitro MN induction by treating TK6 cells with up to 20 concentrations in quadruplicate. In vitro MN frequencies were determined via flow cytometry using a 96-well plate autosampler. The estimated in vitro and in vivo BMDs were found to correlate to each other. The correlation showed considerable scatter, as may be expected given the complexity of the whole animal model versus the simplicity of the cell culture system. Even so, the existence of the correlation suggests that information on the clastogenic potency of a compound can be derived from either whole animal studies or cell culture-based models of chromosomal damage. We also show that the choice of the benchmark response, i.e. the effect size associated with the BMD, is not essential in establishing the correlation between both systems. Our results support the concept that datasets derived from comprehensive genotoxicity studies can provide quantitative dose-response metrics. Such investigational studies, when supported by additional data, might then contribute directly to product safety investigations, regulatory decision-making and human risk assessment.

Flow cytometric evaluation of the contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells.

Exposure to nanosilver found in food- and cosmetics-related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN-forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food- and cosmetics-related chemicals including nanosilver.

Comparative genotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by a flow cytometric in vitro micronucleus assay.

Two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, and flow cytometry techniques were evaluated as tools for rapid screening of potential genotoxicity of food-related nanosilver. Comparative genotoxic potential of 20 nm silver was evaluated in HepG2 and Caco2 cell cultures by a flow cytometric-based in vitro micronucleus assay. The nanosilver, characterized by the dynamic light scattering, transmission electron microscopy and inductively coupled plasma-mass spectrometry analysis, showed no agglomeration of the silver nanoparticles. The inductively coupled plasma-mass spectrometry and transmission electron microscopy analysis demonstrated the uptake of 20 nm silver by both cell types. The 20 nm silver exposure of HepG2 cells increased the concentration-dependent micronucleus formation sevenfold at 10 µg ml(-1) concentration in attached cell conditions and 1.3-fold in cell suspension conditions compared to the vehicle controls. However, compared to the vehicle controls, the 20 nm silver exposure of Caco2 cells increased the micronucleus formation 1.2-fold at a concentration of 10 µg ml(-1) both in the attached cell conditions as well as in the cell suspension conditions. Our results of flow cytometric in vitro micronucleus assay appear to suggest that the HepG2 cells are more susceptible to the nanosilver-induced micronucleus formation than the Caco2 cells compared to the vehicle controls. However, our results also suggest that the widely used in vitro models, HepG2 and Caco2 cells and the flow cytometric in vitro micronucleus assay are valuable tools for the rapid screening of genotoxic potential of nanosilver and deserve more careful evaluation.

Visualization strategies to aid interpretation of high-dimensional genotoxicity data.

This article describes a range of high-dimensional data visualization strategies that we have explored for their ability to complement machine learning algorithm predictions derived from MultiFlow® assay results. For this exercise, we focused on seven biomarker responses resulting from the exposure of TK6 cells to each of 126 diverse chemicals over a range of concentrations. Obviously, challenges associated with visualizing seven biomarker responses were further complicated whenever there was a desire to represent the entire 126 chemical data set as opposed to results from a single chemical. Scatter plots, spider plots, parallel coordinate plots, hierarchical clustering, principal component analysis, toxicological prioritization index, multidimensional scaling, t-distributed stochastic neighbor embedding, and uniform manifold approximation and projection are each considered in turn. Our report provides a comparative analysis of these techniques. In an era where multiplexed assays and machine learning algorithms are becoming the norm, stakeholders should find some of these visualization strategies useful for efficiently and effectively interpreting their high-dimensional data.

In vitro human cell-based aneugen molecular mechanism assay.

This laboratory previously described an in vitro human cell-based assay and data analysis scheme that discriminates common molecular targets responsible for chemical-induced in vitro aneugenicity: tubulin destabilization, tubulin stabilization, and inhibition of Aurora kinases (Bernacki et al., Toxicol. Sci. 170 [2019] 382-393). The current report describes updated procedures that simplify benchtop processing and data analysis methods. For these experiments, human lymphoblastoid TK6 cells were exposed to each of 25 aneugens over a range of concentrations in the presence of fluorescent paclitaxel (488 Taxol). After a 4 h treatment period, cells were lysed and nuclei were stained with a nucleic acid dye and labeled with fluorescent antibodies against phospho-histone H3 (p-H3). Flow cytometric analyses revealed several unique signatures: tubulin stabilizers caused increased frequencies of p-H3-positive events with concentration-dependent increases in 488 Taxol-associated fluorescence; tubulin destabilizers caused increased frequencies of p-H3-positive events with concomitant decreases in 488 Taxol-associated fluorescence; and Aurora kinase B inhibitors caused reduced frequencies of p-H3-positive events and lower median fluorescent intensities of p-H3-positive events. These results demonstrate a simple rubric based on 488 Taxol- and p-H3-associated metrics can reliably discriminate between several commonly encountered aneugenic molecular mechanisms.

When pigs fly: immunomagnetic separation facilitates rapid determination of Pig-a mutant frequency by flow cytometric analysis.

In vivo mutation assays based on the Pig-a null phenotype, that is, the absence of cell surface glycosylphosphatidylinositol (GPI) anchored proteins such as CD59, have been described. This work has been accomplished with hematopoietic cells, most often rat peripheral blood erythrocytes (RBCs) and reticulocytes (RETs). The current report describes new sample processing procedures that dramatically increase the rate at which cells can be evaluated for GPI anchor deficiency. This new method was applied to blood specimens from vehicle, 1,3-propane sultone, melphalan, and N-ethyl-N-nitrosourea treated Sprague Dawley rats. Leukocyte- and platelet-depleted blood samples were incubated with anti-CD59-phycoerythrin (PE) and anti-CD61-PE, and then mixed with anti-PE paramagnetic particles and Counting Beads (i.e., fluorescent microspheres). An aliquot of each specimen was stained with SYTO 13 and flow cytometric analysis was performed to determine RET percentage, RET:Counting Bead ratio, and RBC:Counting Bead ratio. The major portion of these specimens were passed through ferromagnetic columns that were suspended in a magnetic field, thereby depleting each specimen of wild-type RBCs (and platelets) based on their association with anti-PE paramagnetic particles. The eluates were concentrated via centrifugation and the resulting suspensions were stained with SYTO 13 and analyzed on the flow cytometer to determine mutant phenotype RET:Counting Bead and mutant phenotype RBC:Counting Bead ratios. The ratios obtained from pre- and post-column analyses were used to derive mutant phenotype RET and mutant phenotype RBC frequencies. Results from vehicle control and genotoxicant-treated rats are presented that indicate the scoring system is capable of returning reliable mutant phenotype cell frequencies. Using this wild-type cell depletion strategy, it was possible to interrogate ≥ 3 million RETs and ≥ 100 million RBCs per rat in approximately 7 min. Beyond considerably enhancing the throughput capacity of the analytical platform, these blood-processing procedures were also shown to enhance the precision of the measurements.

The use of benchmark dose uncertainty measurements for robust comparative potency analyses.

The Benchmark Dose (BMD) method is the favored approach for quantitative dose-response analysis where uncertainty measurements are delineated between the upper (BMDU) and lower (BMDL) confidence bounds, or confidence intervals (CIs). Little has been published on the accurate interpretation of uncertainty measurements for potency comparative analyses between different test conditions. We highlight this by revisiting a previously published comparative in vitro genotoxicity dataset for human lymphoblastoid TK6 cells that were exposed to each of 10 clastogens in the presence and absence (+/-) of low concentration (0.25%) S9, and scored for p53, γH2AX and Relative Nuclei Count (RNC) responses at two timepoints (Tian et al., 2020). The researchers utilized BMD point estimates in potency comparative analysis between S9 treatment conditions. Here we highlight a shortcoming that the use of BMD point estimates can mischaracterize potency differences between systems. We reanalyzed the dose responses by BMD modeling using PROAST v69.1. We used the resulting BMDL and BMDU metrics to calculate "S9 potency ratio confidence intervals" that compare the relative potency of compounds +/- S9 as more statistically robust metrics for comparative potency measurements compared to BMD point estimate ratios. We performed unsupervised hierarchical clustering that identified four S9-dependent groupings: high and low-level potentiation, no effect, and diminution. This work demonstrates the importance of using BMD uncertainty measurements in potency comparative analyses between test conditions. Irrespective of the source of the data, we propose a stepwise approach when performing BMD modeling in comparative potency analyses between test conditions.

Miniaturized flow cytometric in vitro micronucleus assay represents an efficient tool for comprehensively characterizing genotoxicity dose-response relationships.

This laboratory has developed a flow cytometric approach for scoring in vitro micronuclei (In Vitro MicroFlow(®)) whose characteristics are expected to benefit studies designed to comprehensively investigate genotoxicity dose-response relationships. In particular, new experimental designs become possible when automated scoring is combined with treatment, processing and sampling that all occur in microtiter plates. To test this premise, experiments described herein investigated micronucleus (MN) formation in TK6 cells treated with genotoxic agents applied at 22 closely spaced concentrations in quadruplicate, with 10,000 cells analyzed per replicate. The genotoxicants colchicine, vinblastine sulfate, ethyl methanesulfonate, methyl methanesulfonate, ethyl nitrosourea, methyl nitrosourea, and bleomycin were applied continuously for 24-30 h. Following treatment, all cell processing, sampling and data acquisition steps were accomplished in the same 96-well plate. Data acquisition occurred in a walk-away mode via the use of a high throughput sampling device. The resulting flow cytometric MN values were evaluated with a statistical model that indicated non-linear relationships describe the data better than linear fits. The one exception was bleomycin, where MN induction was consistently best described by a linear dose-response relationship. Collectively, these results suggest that flow cytometry represents a practical and efficient approach for thoroughly examining the dose-response relationship, and clearly benefits studies that seek to characterize no observable genotoxic effect levels, lowest observable genotoxic effect levels, and/or benchmark doses.

3Rs-friendly approach to exogenous metabolic activation that supports high-throughput genetic toxicology testing.

MultiFlow® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/ß-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.

Benchmark Dose Analysis of DNA Damage Biomarker Responses Provides Compound Potency and Adverse Outcome Pathway Information for the Topoisomerase II Inhibitor Class of Compounds.

Genetic toxicology data have traditionally been utilized for hazard identification to provide a binary call for a compound's risk. Recent advances in the scientific field, especially with the development of high-throughput methods to quantify DNA damage, have influenced a change of approach in genotoxicity assessment. The in vitro MultiFlow® DNA Damage Assay is one such method which multiplexes γH2AX, p53, phospho-histone H3 biomarkers into a single-flow cytometric analysis (Bryce et al., [2016]: Environ Mol Mutagen 57:546-558). This assay was used to study human TK6 cells exposed to each of eight topoisomerase II poisons for 4 and 24 hr. Using PROAST v65.5, the Benchmark Dose approach was applied to the resulting flow cytometric datasets. With "compound" serving as covariate, all eight compounds were combined into a single analysis, per time point and endpoint. The resulting 90% confidence intervals, plotted in Log scale, were considered as the potency rank for the eight compounds. The in vitro MultiFlow data showed a maximum confidence interval span of 1Log, which indicates data of good quality. Patterns observed in the compound potency rank were scrutinized by using the expert rule-based software program Derek Nexus, developed by Lhasa Limited. Compound sub-classification and structural alerts were considered contributory to the potencies observed for the topoisomerase II poisons studied herein. The Topo II poison Adverse Outcome Pathway was evaluated with MultiFlow endpoints serving as Key Events. The step-wise approach described herein can be considered as a foundation for risk assessment of compounds within a specific mode of action of interest. Environ. Mol. Mutagen. 2020. © 2020 Wiley Periodicals, Inc.

Application of the adverse outcome pathway framework to genotoxic modes of action.

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114-134, 2020. © 2019 Wiley Periodicals, Inc.

Aneugen Molecular Mechanism Assay: Proof-of-Concept With 27 Reference Chemicals.

A tiered bioassay and data analysis scheme is described for elucidating the most common molecular targets responsible for chemical-induced in vitro aneugenicity: tubulin destabilization, tubulin stabilization, and inhibition of mitotic kinase(s). To evaluate this strategy, TK6 cells were first exposed to each of 27 presumed aneugens over a range of concentrations. After 4 and 24 h of treatment, γH2AX, p53, phospho-histone H3 (p-H3), and polyploidization biomarkers were evaluated using the MultiFlow DNA Damage Assay Kit. The assay identified 27 of 27 chemicals as genotoxic, with 25 exhibiting aneugenic signatures, 1 aneugenic and clastogenic, and 1 clastogenic. Subsequently, a newly described follow-up assay was employed to investigate the aneugenic agents' molecular targets. For these experiments, TK6 cells were exposed to each of 26 chemicals in the presence of 488 Taxol. After 4 h, cells were lysed and the liberated nuclei and mitotic chromosomes were stained with a nucleic acid dye and labeled with fluorescent antibodies against p-H3 and Ki-67. Flow cytometric analyses revealed that alterations to 488 Taxol-associated fluorescence were only observed with tubulin binders-increases in the case of tubulin stabilizers, decreases with destabilizers. Mitotic kinase inhibitors with known Aurora kinase B inhibiting activity were the only aneugens that dramatically decreased the ratio of p-H3-positive to Ki-67-positive nuclei. Unsupervised hierarchical clustering based on 488 Taxol fluorescence and p-H3: Ki-67 ratios clearly distinguished compounds with these disparate molecular mechanisms. Furthermore, a classification algorithm based on an artificial neural network was found to effectively predict molecular target, as leave-one-out cross-validation resulted in 25/26 agreement with a priori expectations. These results are encouraging, as they suggest that an adequate number of training set chemicals, in conjunction with a machine learning algorithm based on 488 Taxol, p-H3, and Ki-67 responses, can reliably elucidate the most commonly encountered aneugenic molecular targets.

Evidence for an Aneugenic Mechanism of Action for Micronucleus Induction by Black Cohosh Extract.

Black cohosh extract (BCE) is a popular botanical dietary supplement marketed to relieve symptoms of various gynecological ailments. Studies conducted by the National Toxicology Program (NTP) showed that BCE induces micronucleated erythrocytes in female rats and mice. Subsequently, the NTP showed that a variety of BCEs, including the sample that induced micronuclei (MN) in vivo ("NTP BCE") had a similar effect in human TK6 cells. Further testing with the MultiFlow® DNA Damage Assay revealed that TK6 cells exposed to NTP BCE, as well as a BCE reference material (BC XRM), exhibited a signature consistent with aneugenic activity in TK6 cells. Results from experiments reported herein confirmed these in vitro observations with NTP BCE and BC XRM. We extended these studies to include a novel test system, the MultiFlow Aneugen Molecular Mechanism Assay. For these experiments, TK6 cells were exposed to NTP BCE and BC XRM over a range of concentrations in the presence of fluorescent Taxol (488 Taxol). After 4 h, nuclei from lysed cells were stained with a nucleic acid dye and labeled with fluorescent antibodies against phospho-histone H3 (p-H3) and Ki-67. Whereas BCEs did not affect p-H3:Ki-67 ratios (a signature of aneugenic mitotic kinase inhibitors), 488 Taxol-associated fluorescence (a tubulin binder-sensitive endpoint) was affected. More specifically, 488 Taxol-associated fluorescence was reduced over the same concentration range that was previously observed to induce MN. These results provide direct evidence that BCEs destabilize microtubules in vitro, and this is the molecular mechanism responsible for the aneugenicity findings. Environ. Mol. Mutagen. 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Predictions of genotoxic potential, mode of action, molecular targets, and potency via a tiered multiflow® assay data analysis strategy.

The in vitro MultiFlow® DNA Damage Assay multiplexes γH2AX, p53, phospho-histone H3, and polyploidization biomarkers into a single flow cytometric analysis. The current report describes a tiered sequential data analysis strategy based on data generated from exposure of human TK6 cells to a previously described 85 chemical training set and a new pharmaceutical-centric test set (n = 40). In each case, exposure was continuous over a range of closely spaced concentrations, and cell aliquots were removed for analysis following 4 and 24 hr of treatment. The first data analysis step focused on chemicals' genotoxic potential, and for this purpose, we evaluated the performance of a machine learning (ML) ensemble, a rubric that considered fold increases in biomarkers against global evaluation factors (GEFs), and a hybrid strategy that considered ML and GEFs. This first tier further used ML output and/or GEFs to classify genotoxic activity as clastogenic and/or aneugenic. Test set results demonstrated the generalizability of the first tier, with particularly good performance from the ML ensemble: 35/40 (88%) concordance with a priori genotoxicity expectations and 21/24 (88%) agreement with expected mode of action (MoA). A second tier applied unsupervised hierarchical clustering to the biomarker response data, and these analyses were found to group certain chemicals, especially aneugens, according to their molecular targets. Finally, a third tier utilized benchmark dose analyses and MultiFlow biomarker responses to rank genotoxic potency. The relevance of these rankings is supported by the strong agreement found between benchmark dose values derived from MultiFlow biomarkers compared to those generated from parallel in vitro micronucleus analyses. Collectively, the results suggest that a tiered MultiFlow data analysis pipeline is capable of rapidly and effectively identifying genotoxic hazards while providing additional information that is useful for modern risk assessments-MoA, molecular targets, and potency. Environ. Mol. Mutagen. 60:513-533, 2019. © 2019 Wiley Periodicals, Inc.

In vivo mutation assay based on the endogenous Pig-a locus.

The product of the X-chromosome's Pig-a gene acts in the first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis, and is thereby essential for attaching certain proteins to the cell surface. The experiments described herein were designed to evaluate whether lack of GPI-anchored proteins could form the basis of an in vivo mutation assay. Specifically, we used a CD59-negative cell surface phenotype to denote Pig-a mutation. Besides anti-CD59-PE, two other fluorescent reagents were used: thiazole orange to differentiate mature erythrocytes, reticulocytes (RETs), and leukocytes; and anti-CD61 to resolve platelets. These experiments were performed with Sprague Dawley rats, and focused on two cell populations, total erythrocytes and RETs. The ability of the analytical method to enumerate CD59-negative erythrocytes was initially assessed with reconstruction experiments whereby mutant-mimicking cells were added to control bloods. Subsequently, female rats were treated on three occasions with the model mutagens ENU (100 mg/kg/day) or DMBA (40 mg/kg/day). Blood specimens were harvested at various intervals, as late as 6 weeks post-exposure. Considering all week 4-6 data, we found that CD59-negative cells ranged from 239 to 855 x 10(-6) and 82 to 405 x 10(-6) for ENU and DMBA, respectively. These values were consistently greater than those observed for negative control rats (18 +/- 19 x 10(-6)). The elevated frequencies observed for the genotoxicant-exposed animals were usually higher for RETs compared to total erythrocytes. These data support the hypothesis that an efficient in vivo mutation assay can be developed around flow cytometric enumeration of erythrocytes and/or RETs that exhibit aberrant GPI-anchored protein expression.

Erythrocyte-based Pig-a gene mutation assay: demonstration of cross-species potential.

Glycosylphosphatidylinositol (GPI) anchors attach specific proteins to the cell surface of hematopoietic cells. Of the genes required to form GPI anchors, only Pig-a is located on the X-chromosome. Prior work with rats suggests that the GPI anchor deficient phenotype is a reliable indicator of Pig-a mutation [Bryce et al., Environ. Mol. Mutagen., 49 (2008) 256-264]. The current report extends this line of investigation by describing simplified blood handling procedures, and by testing the assay principle in a second species, Mus musculus. With this method, erythrocytes are isolated, incubated with anti-CD24-PE, and stained with SYTO 13. Flow cytometric analyses quantify GPI anchor-deficient erythrocytes and reticulocytes. After reconstruction experiments with mutant-mimicking cells demonstrated that the analytical performance of the method is high, CD-1 mice were treated on three occasions with 7,12-dimethyl-1,2-benz[a]anthracene (DMBA, 75 mg/kg/day) or ethyl-N-nitrosourea (ENU, 40 mg/kg/day). Two weeks after the final treatment, DMBA-treated mice were found to exhibit markedly elevated frequencies of GPI anchor deficient erythrocytes and reticulocytes. For the ENU experiment, blood specimens were collected at weekly intervals over a 5-week period. Whereas the frequencies of mutant reticulocytes were significantly elevated 1 week after the last administration, the erythrocyte population was unchanged until the second week. Thereafter, both populations exhibited persistently elevated frequencies for the duration of the experiment (mean frequency at termination=310x10(-6) and 523x10(-6) for erythrocyte and reticulocyte populations, respectively). These data provide evidence that Pig-a mutation does not convey an appreciable positive or negative cell survival advantage to affected erythroid progenitors, although they do suggest that affected erythrocytes have a reduced lifespan in circulation. Collectively, accumulated data support the hypothesis that flow cytometric enumeration of GPI anchor deficient erythrocytes and/or reticulocytes represents an effective in vivo mutation assay that is applicable across species of toxicological interest.

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay.

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Mol. Mutagen. 47 (2006) 56-66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei -- the genotoxicants mitomycin C (MMC), etoposide (ETOPO), and vinblastine (VB), and the non-genotoxicants sucrose (SUC), staurosporine (STS), and dexamethasone (DEX). The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24h over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.

Investigating the Generalizability of the MultiFlow ® DNA Damage Assay and Several Companion Machine Learning Models With a Set of 103 Diverse Test Chemicals.

The in vitro MultiFlow DNA Damage assay multiplexes p53, γH2AX, phospho-histone H3, and polyploidization biomarkers into 1 flow cytometric analysis (Bryce, S. M., Bernacki, D. T., Bemis, J. C., and Dertinger, S. D. (2016). Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach. Environ. Mol. Mutagen. 57, 171-189). The work reported herein evaluated the generalizability of the method, as well as several data analytics strategies, to a range of chemical classes not studied previously. TK6 cells were exposed to each of 103 diverse chemicals, 86 of which were supplied by the National Toxicology Program (NTP) and selected based upon responses in genetic damage assays conducted under the Tox21 program. Exposures occurred for 24 h over a range of concentrations, and cell aliquots were removed at 4 and 24 h for analysis. Multiplexed response data were evaluated using 3 machine learning models designed to predict genotoxic mode of action based on data from a training set of 85 previously studied chemicals. Of 54 chemicals with sufficient information to make an a priori call on genotoxic potential, the prediction models' accuracies were 79.6% (random forest), 88.9% (logistic regression), and 90.7% (artificial neural network). A majority vote ensemble of the 3 models provided 92.6% accuracy. Forty-nine NTP chemicals were not adequately tested (maximum concentration did not approach assay's cytotoxicity limit) and/or had insufficient conventional genotoxicity data to allow their genotoxic potential to be defined. For these chemicals MultiFlow data will be useful in future research and hypothesis testing. Collectively, the results suggest the MultiFlow assay and associated data analysis strategies are broadly generalizable, demonstrating high predictability when applied to new chemicals and classes of compounds.

Comparative Genotoxicity of TEMPO and 3 of Its Derivatives in Mouse Lymphoma Cells.

TEMPO (2, 2, 6, 6-tetramethylphiperidine-1-oxyl) and its derivatives are stable free radical nitroxides widely used in the field of chemistry, biology, and pharmacology. TEMPO was previously found to be mutagenic and to induce micronuclei in mammalian cells. In this study, we investigated and quantified the genotoxicity of 4 structurally similar nitroxides, TEMPO and 3 of its derivatives (4-hydroxy-TEMPO, 4-oxo-TEMPO, and 4-methoxy-TEMPO), using the mouse lymphoma assay (MLA) and Comet assay in L5178Y Tk+/- cells. The results showed that all tested nitroxides were cytotoxic and mutagenic in the MLA, both in the presence and absence of S9, with metabolic activation significantly enhancing the cytotoxicity and/or mutagenicity. In addition, the 4 nitroxides caused DNA-strand breakage. The mutagenicity and DNA damaging dose-responses of the test articles were compared using the PROAST benchmark dose software package. The potency ranking of the 4 nitroxides for mutagenicity was different from the ranking of the DNA damaging effects. The mode of action analysis by a multi-endpoint DNA damage pathway assay classified all 4 nitroxides as clastogens. In addition, the majority of the induced Tk mutants showed loss of heterozygosity at the Tk and D11Mit42 loci (ie, chromosome damage <31 Mbp). These results suggest that TEMPO and its 3 derivatives are cytotoxic and mutagenic in mouse lymphoma cells through a mechanism that involves strand breakage and large alterations to DNA. The potency rankings indicate that the different TEMPO derivatives vary in their mutagenic and DNA damaging potential.